EXERCISE 20. EFFECT OFTEMPERATURE ON GROWTH OF

MICROORGANISMS

EFFECT OF TEMPERATURE ON GROWTH OF MICROORGANISMS

Cardinal temperaturesMinimum temperature: : It is the lowest temperature which will support the growth of the microorganism. Below the minimum temperature the microorganismssurvive for a long time and such low temperatures may be used for their storage.

Optimal temperature: It is the temperature, or more usually the range of temperatures, which supports thefastest growth rate.

Maximum temperature: It is the highest temperature which will support the growth of the microorganism. Above the maximum temperature the microorganismis usually killed and such high temperatures may be used in sterilization.

Types of microorganisms classified by optimum temperature:

• Psychrophiles:• Psychrotrophs (facultative psychrophiles):• Mesophiles:• Thermophiles:• Thermotolerant and thermoduric microorganisms:• Hyperthermophiles:

Types of microorganisms classified by optimum temperature:

Adaptations to cold temperatures

• Membrane composition (increased membrane fluidity)

– Increased number of double bonds in membrane fatty acids

– Increased amount of hopanoids (in bacteria) or cholesterol (in eukarya) in membrane

– Decreased fatty acid chain length• Enzymes– Must function efficiently at

lower temperatures

Adaptations to high temperatures

• Membrane composition– Decreased number of double bonds in membrane fatty

acids– Decreased amount of hopanoids or cholesterol– Increased fatty acid chain length– Archaea – use ether linkages instead of ester linkages –

greater stability at hightemperatures.• Some archaea use tetraether lipids that form a monolayer

instead of a lipid bilayer.• Proteins and enzymes– More cross-linking or altered composition (change the

hydrophilic and hydrophobicinteractions, increased charge interactions between amino

acids) for greater proteinstability• Ribosomes and other molecular machines need to be

very stable

MethodSerratia marcescens and Escherichia coli

1. Label the tubes as followsSlants: label one 25oC (RT), and the

other 37oC,Broths: 5oC, 25oC (RT), and 42oC2. Inoculate the tubes as followsSlants: one with E. coli, other one

with S. marcescensBroths: E. coli or S. marcescens3. Place the tubes in racks and

incubate at proper temperatures for 24 hours.

Evaluation:Slants: Compare the nutrient agar slants of

S. marcescens. Using colored pencils, draw

the appearance of the growths on the laboratory report.

Broths:1. Calibrate the spectrophotometer with

sterile nutrient broth according to theinstructor’s direction.2. Shake each culture sufficiently to

completely disperse the organisms and pour 2

ml into cuvette in given order.3. Measure the absorbance (O.D.) of your

cultures and record these values on thelaboratory report.4. Plot the O.D. values on the graph-paper.

EXERCISE 21. LETHAL EFFECT OFTEMPERATURE

The factors affecting the heat resistance of a microorganism:

• The species and strain,• How it is grown,• The medium in which it is heated (food vs.

buffer), • The environment in which it will recover after

heating.

Measurements used in order to compare the susceptibility of different organisms to increased

temperatures• Thermal Death Point (TDP): lowest temperature

required to kill all microbes in liquid suspension in 10 minutes.

• Thermal Death Time (TDT): Minimal time required to kill all microorganisms keeping temperature (or bacteriocidal agent) constant; i.e. at a specific temperature.

• Decimal Reduction Time (DRT or D value): Used for heat resistant bacteria; Time required to kill 90% of the population at a given temperature (Log growth

and log death concept).* Moisture, pH, composition of medium and age of cells will greatly influence these values

Materials:1. 24 h nutrient broth culture

of the following organisms:a. Escherichia coli DH5αb. Bacillus thuringiensis2. Nutrient agar plates (five for

each group)

Method:• Label five agar plates as; control, 10 min., 20 min., 30min., and

40 min.,respectively.• Each group is assigned one culture of organisms to be heated at

a specific temperature (55-65-75-85 oC)• Shake the culture of organisms and transfer 0.1 ml of the

organism with a 1 mlpipette to control plate.• Place the culture of your group into the water bath.• As soon as the temperature reaches the desired one, record the

time. Watch the temperature carefully to make sure it does not vary appreciably.

• After ten minutes have elapsed, transfer 0.1 ml from the culture to the 10 min. plate with a fresh 1 ml pipette. Repeat this operation at ten minute intervals until

all the plates that have been inoculated. Use fresh pipettes each time and be sure to shake the culture.

• Spread the culture over the medium by rotating the glass spreader.

• Incubate at 37oC for 24 – 48 hours.

Evaluation

EXERCISE 22. OSMOTIC PRESSUREAND BACTERIAL GROWTH

• Osmosis: the net movement of H2O across a semipermeable membrane (i.e. cell membrane).

• Osmotic pressure: the force that drives osmosis. It is caused by different solute concentrations in the cell and its environment.

Osmophiles– Love high osmotic pressure– Salts or sugarsSaccharophiles:– Osmophiles which love high sugar Halophiles:– Osmophiles which love high saltXerophiles:– Osmophiles which love very dryenvironments

Material1. 24 h nutrient broth cultures of

following organisms:a. Escherichia colib. Staphylococcus aureusc. Bacillus subtilis2. Following plates for each group:• 1 nutrient agar plate (0.5 %

NaCl)• 1 nutrient agar plate (5 % NaCl)• 1 nutrient agar plate (10 % NaCl)• 1 nutrient agar plate (15 % NaCl)

Method1. Mark the bottoms of the four Petri plates as indicated in below2. Streak each organism in a straight line on the media, using a wire loop.3. Incubate all the plates at 37oC for 48 h

Evaluation